Synthesis and SAR study of new hydroxy and chloro-substituted 2,4-diphenyl 5H-chromeno[4,3-b]pyridines as selective topoisomerase IIα-targeting anticancer agents

Article abstract of DOI:10.1016/j.bmc.2018.02.035

As part of our effort to develop potential topoisomerase IIα (topo IIα) targeting anticancer agents, we systematically designed a new series of hydroxy and chloro-substituted 2,4-diphenyl 5H-chromeno[4,3-b]pyridines. Total eighteen compounds were synthesi

Full text of DOI:10.1016/j.bmc.2018.02.035

Accepted Manuscript
Synthesis and SAR study of new hydroxy and chloro-substituted 2,4-diphenyl
5
H-chromeno[4,3-b]pyridines as selective topoisomerase IIα-targeting anti-
cancer agents
Til Bahadur Thapa Magar, Seung Hee Seo, Tara Man Kadayat, Hyunji Jo,
Aarajana Shrestha, Ganesh Bist, Pramila Katila, Youngjoo Kwon, Eung-Seok
Lee
PII:
S0968-0896(18)30113-5
https://doi.org/10.1016/j.bmc.2018.02.035
BMC 14221
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
18 January 2018
19 February 2018
20 February 2018
Please cite this article as: Magar, T.B.T., Seo, S.H., Kadayat, T.M., Jo, H., Shrestha, A., Bist, G., Katila, P., Kwon,
Y., Lee, E-S., Synthesis and SAR study of new hydroxy and chloro-substituted 2,4-diphenyl 5H-chromeno[4,3-
b]pyridines as selective topoisomerase IIα-targeting anticancer agents, Bioorganic & Medicinal Chemistry (2018),
doi: https://doi.org/10.1016/j.bmc.2018.02.035
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Synthesis and SAR study of new hydroxy and chloro-substituted 2,4-diphenyl 5H-
chromeno[4,3-b]pyridines as selective topoisomerase IIα-targeting anticancer agents
a,1
b,1
a,1
b
†
Til Bahadur Thapa Magar , Seung Hee Seo , Tara Man Kadayat , Hyunji Jo , Aarajana
a
a
a
b,*
a,*
Shrestha , Ganesh Bist , Pramila Katila , Youngjoo Kwon , Eung-Seok Lee
a
b
College of Pharmacy, Yeungnam University, Gyeongsan 38541, Republic of Korea
College of Pharmacy, Graduate School of Pharmaceutical Sciences, Ewha Womans
University, Seoul 120-750, Republic of Korea
*
Corresponding Authors:
email: ykwon@ewha.ac.kr (Y.K.)
email: eslee@yu.ac.kr (E.-S.L.)
1
Authors are equal contributors
†Present Address: New Drug Development Center, Daegu-Gyeongbuk Medical Innovation
Foundation, Daegu 41061, Republic of Korea
1

Synthesis and SAR study of new hydroxy and chloro-substituted 2,4-diphenyl 5H-
chromeno[4,3-b]pyridines as selective topoisomerase IIα-targeting anticancer agents
a,1
b,1
a,1
b
†
Til Bahadur Thapa Magar , Seung Hee Seo , Tara Man Kadayat , Hyunji Jo , Aarajana
a
a
a
b,*
a,*
Shrestha , Ganesh Bist , Pramila Katila , Youngjoo Kwon , Eung-Seok Lee
Abstract
As part of our effort to develop potential topoisomerase IIα (topo IIα) targeting anticancer
agents, we systematically designed a new series of hydroxy and chloro-substituted 2,4-
diphenyl 5H-chromeno[4,3-b]pyridines. Total eighteen compouds were synthesized and
tested for their ablity to inhibit the function of topo I and IIα, and proliferation of human
breast (T47D), colorectal (HCT15), and cervix (HeLa) cancer cells. Except compound 11, all
of the tested compounds displayed selective topo IIα inhibitory activity. Compounds 8-18, 22,
2
4, and 25 showed excellent topo IIα inhibitory activity than a positive control, etoposide.
Most of the compounds appeared to be superior to reference compounds in their
antiproliferative activity. Structure-activity relationship (SAR) study has shown that it is
better to place the hydroxyphenyl group at the 4-position of the central pyridine for superior
topo IIα inhibition and antiproliferative activity. Similarly, the 3′-, or 4′-hydroxyphenyl
substitution at the 2- and 4-positon of pyridine ring is important for better activity than 2′-
substitution.
Keywords: Anticancer agents; 5H-chromeno[4,3-b]pyridines; hydroxyl and chlorine-
substitution; Selective topoisomerase IIα inhibition; SAR study
2

1
. Introduction
In all prokaryotic and eukaryotic life, cell division is the only means of multiplication
and the survival. Cell multiplication proceeds by forming daughter DNA from parental DNA
1
through important cellular processes such as DNA transcription and replication. During
these cellular processes, the DNA helix tangles and bends to form a positive or negative
supercoil. Topoisomerases (topos) are enzymes that solve the topological problems of DNA
by converting the supercoiled DNA to relaxed form allowing vital cellular processes to
2
proceed further. There are two types of topo, depending upon their capabilities: a) topo I that
cleaves the single strand of DNA at a time; and b) topo II that simultaneously breaks the
3
, 4
double strand of DNA.
Since topo II plays major role during cell proliferation, many
researchers are focused to develop safe compounds that inhibit topo II function. Among
different phases of the cell cycle, the synthesis phase (S phase) is considered to be the most
important at which DNA gets duplicated. S phase lies in between G and G phases followed
1
2
5
by mitotic phase (M phase) when the mother cell divides into two daughter cells. Several
studies showed that the level of alpha isoform of topo II (topo IIα) increases significantly at S
phase and reaches maximum at G /M phase while level of another isoform, topo IIβ, remains
2
6
-8
constant throughout the cell cycle. Therefore, inhibition of the topo IIα function results in
interruption at vital stage of cell cycle, consequently leading to cell death.
Chromene is considered an important moiety in medicinal chemistry. Several chromene-
9
-11
12
derived compounds have shown biological activities such as anticancer,
antifungal,
1
3
14
15
16
antimicrobial,
anti-inflammatory,
antiproliferative,
and antiangiogenic activity.
Chromenopyridine is a three-ring system containing heterocyclic moiety which is formed by
the fusion of a chromene and a pyridine fraction. Recently, four different compounds having
chromenopyridine moiety were isolated from a fungus Phomopsis sp. possessing moderate
3

nitric oxide inhibitory activity in LPS induced RAW 264.7 macrophage and good antioxidant
1
7
activity. However, to date the major sources of compounds with chromenopyridine
scaffolds are synthetic approach and different types of reaction methods have been utilized
for their easier synthesis. Chromenopyridine skeleton have attracted many researchers for the
discovery of new drug candidate due to their ability to adjust activity and selectivity via
altering substitution pattern at its different positions.
Previously, our group discovered chromenopyridine derivatives possessing aryl moieties
1
8
such as thienyl, furyl, pyridyl and phenyl as new and potential anticancer agents. Moreover,
we reported hydroxylated chromenopyridines exhibiting selective topo IIα inhibitory activity
1
9
as well as moderate antiproliferative activity (Figure 1A). Several studies have reported that
halogen and hydroxyl group containing compounds possess better biological activity due to
their ability to form of halogen bond and hydrogen bond, respectively, with active site of
2
0-22
receptor.
Recently, we reported 2-chlorophenly-4-phenol-benzofuro[3,2-b]pyridines (A-I)
containing constrained five-membered heterocycle (furan), which displayed excellent dual
topo I/IIα inhibition and effectively suppressed the proliferation of human colorectal
23
adenocarcinoma (HCT15) cell line. In our quest to search novel topo IIα-targeted anticancer
agents, we decided to further design and synthesize a new series of hydroxy and chloro-
substituted 2,4-diphenyl 5H-chromeno[4,3-b]pyridines by ring expansion from 5-membered
furan ring to the six-membered pyran ring (Figure 1B). In addition, we investigated their topo
inhibitory activity as well as antiproliferative activity. Herein, in this study we report design,
synthesis and structure-activity relationships of 5H-chromeno[4,3-b]pyridine derivatives as
novel topo IIα-targeted anticancer agents.
4

Figure 1. Chemical structure of (A) reported bioactive chromenopyridines, and (B) our
designed compounds.
5

2
. Results and discussion
2
.1. Chemistry
The target compounds 8-25 were synthesized from intermediates 5a-5f and pyridinium
iodide salts 7 (R = a-f). Scheme 1 illustrates the general synthetic route for the synthesis of 4-
chromanone intermediates (5a-5f) and pyridinium iodide salts 7 (R = a-f). Intermediates 5a
and 5b were synthesized by protecting hydroxyl moiety of corresponding aldehydes (1a and
1
b) with chloromethyl methyl ether (MOMCl) in the presence of N,N-diisopropylethylamine
(DIPEA). Then, resulting hydroxyl protected aldehydes 2a and 2b were condensed with 4-
chromanone (3) in basic condition to give products 4a and 4b. Deprotection was achieved by
refluxing with aqueous 2 M HCl in ethanol to afford 5a and 5b. Para-hydroxyl containing
intermediate (5c) was obtained by acid catalyzed condensation of 4-hydroxybenzaldehyde (1c)
and 4-chromanone (3) in the presence of boron trifluoride etherate [BF O(C H ) ] in dioxane.
3
2
5 2
Secondly, pyridinium iodide salts 7 (R = a-f) were prepared by refluxing equivalent amount
of corresponding aryl methyl ketone 6 (R = a-f) with iodine in pyridine. Finally, modified
Kröhnke pyridine synthetic method facilitated the reaction of pyridinium iodide salts 7 (R= a-
f) and intermediates 5a-5f in glacial acetic acid using NH OAc as an ammonia source. The
4
reaction was performed under reflux condition for 12-24 h to obtain total eighteen hydroxy
and chloro-substituted 2,4-diphenyl 5H-chromeno[4,3-b]pyridines as final products at the
yield ranging from 22.1% to 74.3% (Scheme 2). Figure 2 shows the structures of target final
compounds 8-25. Physicochemical characterization data of all prepared compounds, such as,
purity (%) as determined by HPLC, melting point (°C), and yield (%), are given in
Supplementary Data.
6

Scheme 1. Synthesis of 4-chromanone intermediates 5a-5f and aryl pyridinium iodide salts
a-7f. Reagents and conditions: (i) MOMCl (1.5 equiv.), DIPEA (3.0 equiv.), CH Cl , 3 h,
7
2
2
room temperature, 86.3-90.1% yield; (ii) aq. NaOH, methanol, 24 h, room temperature, 74.0-
5.6% yield; (iii) aq. 2 M HCl (1.5 equiv.), 2 h, 80 °C, 50.2-50.5% yield; (iv) BF Et O (1.0
9
3
.
2
equiv.), dioxane, 24 h, room temperature, 56.6% yield; (v) aq. NaOH, ethanol, 1-3 h, room
temperature, 61.3-81.8% yield; (vi) iodine (1.0 equiv.), pyridine, 3 h, 140 °C, 52.2-93.0%
yield.
7

Scheme 2. Synthesis of hydroxy and chloro-substituted 2,4-diphenyl 5H-chromeno[4,3-
b]pyridines 8-25. Reagents and conditions: (i) NH OAc (10.0 equiv.), glacial acetic acid, 12-
4
2
4 h, 100 °C, 22.1-74.3% yield.
8

Figure 2. Structures of the synthesized hydroxy and chloro-substituted 2,4-diphenyl 5H-
chromeno[4,3-b]pyridines.
9

2
.2. Topoisomerase I and IIα inhibitory activity
We investigated the ability of topo I and IIα to transform the supercoiled plasmid DNA to
relaxed form under the influence of synthesized compounds (8-25). Two different
concentrations, 100 µM and 20 µM of those compounds were used. Only those compounds
displaying more than 30% inhibition at 100 µM were selected for evaluation in 20 µM
concentration. Clinically available anticancer drugs, camptothecin as a selective topo I
inhibitor and etoposide as a selective topo II inhibitor, were utilized as positive controls. The
extent of topo I and IIα inhibition for the evaluated compounds is compiled in Table 1 and
Figure 3.
2
.2.1. Topoisomerase I inhibitory activity
Majority of the tested compounds showed weak topo I inhibitory activity as compared to
a reference compound, camptothecin, at both 100 µM and 20 µM with exception of
compound 11. Compound 11 possessing 2′-chlorophenyl at 2-position and 3′-hydroxyphenyl
at 4-position of central pyridine ring displayed the most potent topo I inhibitory activity
(79.1%) at 100 µM which is comparable to the positive control, camptothecin (79.9%).
Compound 19 showed considerable topo I inhibition (56.1%) at 100 µM concentration (Table
1
, Figure 3A).
2
.2.2. Topoisomerase IIα inhibitory activity
Table 1 and Figure 3B demonstrate the topo IIα inhibitory activities of the synthesized
compounds 8-25. Most of the evaluated compounds displayed comparable to significant topo
IIα inhibition at both 100 µM and 20 µM concentrations. All the compounds 8-16, containing
2
′-, 3′-, or 4′-chlorophenyl moiety at 2-position and 2′-, 3′-, or 4′-hydroxyphenyl moiety at 4-
position displayed better topo IIα inhibitory activity (71.3-95.9% at 100 µM and 41.9-74.5%
1
0

at 20 µM) than etoposide (66.5% at 100 µM and 35.5% at 20 µM), respectively. Among them,
compound 15 significantly inhibited topo IIα activity at both 100 µM and 20 µM
concentrations (95.9% at 100 µM and 57.3% at 20 µM). Similarly, compound 14 having 2′-
chlorophenyl at 2-position and 4′-hydroxyphenyl at 4-position exhibited two-fold more potent
as compared with reference compound, etoposide, at 20 µM. On the other hand, compounds
1
7-25 possessing 2′-, 3′-, or 4′-hydroxyphenyl moiety at 2-position, and 2′-, 3′-, or 4′-
chlorophenyl moiety at 4-position showed comparable to strong topo IIα inhibition. Among
them, compounds 17, 18, 22, 24 and 25 displayed better activity (66.7-100%) than reference
compound at 100 µM. Compound 25 showed 100% topo IIα inhibition at 100 µM.
1
1

(A)Topo I inhibition
(B) Topo IIα inhibition
Figure 3. DNA topo I (A) and topo IIα (B) inhibitory activity of the synthesized compounds
-25 at the concentrations of 100 µM and 20 µM. (A) Lane D: pBR322 only, Lane T:
pBR322 + Topo I, Lane C: pBR322 + Topo I + Camptothecin, Lane 8-25: pBR322 + Topo I
compounds 8-25 at 100 μM or 20 μM. (B) Lane D: pBR322 only, Lane T: pBR322 + Topo
IIα, Lane E: pBR322 + Topo IIα + Etoposide, Lane 8-25: pBR322 + Topo IIα + compounds
-25 at 100 μM or 20 μM.
8
+
8
1
2

Table 1. Topo I and IIα inhibitory and antiproliferative activity of the prepared compounds 8-
5.
2
Compounds
%Inhibition
*IC (μM)
50
Topo IIα
Topo I
HCT15
T47D
HeLa
1
00 μM 20 μM 100 μM 20 μM
Camptothecin
79.9
34.3
0.02±0.00 0.49±0.02 0.13±0.00
2.45±0.08 0.32±0.01 10.03±0.33
0.73±0.01 0.84±0.01 1.70±0.06
Etoposide
66.5
35.5
Adriamycin
8
9
79.1
75.1
88.9
90.8
84.0
71.3
82.7
95.9
87.8
78.1
91.7
63.5
18.9
63.6
79.5
34.7
66.7
100.0
47.3
51.9
56.6
57.3
63.7
63.8
74.5
57.3
41.9
45.5
57.0
48.1
NT
4.7
4.4
NT
NT
NT
1.7
NA
NT
NT
NT
NT
NT
NT
NA
NT
NT
NT
NT
NT
1.7
>50
2.23±0.08 1.23±0.04 0.68±0.01
1.66±0.03 >50 >50
>50
>50
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
7.6
79.1
30.0
28.8
NA
0.87±0.01 0.70±0.01 2.50±0.10
2.13±0.09 0.72±0.02 0.68±0.02
2.30±0.39 1.18±0.01 0.76±0.00
1.74±0.05 0.75±0.01 2.11±0.01
1.77±0.02 0.88±0.08 0.89±0.01
1.83±0.06 1.72±0.14 0.86±0.03
NA
NA
NA
>50
1.66±0.05
>50
NA
1.52±0.05 0.86±0.01 0.74±0.03
4.72±0.12 0.94±0.13 0.68±0.01
56.1
21.1
12.0
22.8
20.1
20.2
38.5
>50
>50
>50
NA
1.23±0.01 0.86±0.01 0.82±0.01
3.41±0.55 1.27±0.01 0.66±0.01
12.1
NA
>50
>50
>50
4.7
4.57±0.18 0.75±0.01 1.94±0.06
1.85±0.03 1.38±0.03 2.08±0.24
19.9
NT: not tested; NA; not active.
HCT15: human colorectal adenocarcinoma cell line; T47D: human breast cancer cell line;
HeLa: human cervix tumor cell line. Camptothecin: positive control for topo I and
antiproliferative activity; Etoposide: positive control for topo IIα and antiproliferative
activity; Adriamycin: positive control for antiproliferative activity.
*Results represent mean ± S.D. from three different experiments performed in triplicate.
1
3

2
.3. Antiproliferative activity
Table 1 displays the antiproliferative activity (IC ) of the synthesized compounds 8-25
50
against three different cancer cell lines: HCT15 (human colorectal adenocarcinoma cell line),
T47D (human breast cancer cell line), and HeLa (human cervix tumor cell line). Except
compounds 8, 10, 17, 20, and 23, all other compounds displayed significantly better
antiproliferative activity (0.66 µM to 2.08 µM) than etoposide (10.03 µM) against HeLa
cancer cell line. Comparatively, compound 11 possessing 2′-chlorophenyl at 2-position and
3
′-hydroxyphenyl at 4-position of central pyridine ring, which showed significant dual topo I
and IIα inhibitory activity displayed the most potent antiproliferative activity against HCT15
0.87 µM) and T47D (0.70 µM). Similarly, compound 22 possessing 4′-hydroxyphenyl
(
moiety at 2-position and 3′-chlorophenyl moiety at 4-position which displayed a selective
topo IIα inhibition, showed 15.2 and 2.6 fold more strong antiproliferative activity against
HeLa cell lines (0.66 µM) than reference compounds, etoposide and adriamycin, respectively.
Compounds 11, 12, 14, 15, 18, 19, 21, and 24 have demonstrated comparable to stronger
antiproliferative activity (0.70 µM to 0.94 µM) against T47D as compared with adriamycin
(0.84 µM). It was observed that most of the compounds exhibiting significant topo IIα
inhibition also displayed comparable to significant antiproliferative activity as compared with
positive controls, etoposide and adriamycin, reflecting that there was positive correlation
between selective topo IIα inhibition and antiproliferative activity.
1
4

2
.4. Structure-activity relationship study (SAR)
In this study, most of the compounds with 2′-, 3′-, or 4′-hydroxyphenyl group at 4-
position (8-16) exhibited better topo IIα inhibitory activity as well as antiproliferative activity
than compounds containing 2′-, 3′-, or 4′-hydroxyphenyl group at 2-position (17-25) of
central pyridine ring. This results indicated the importance of hydroxyphenyl moiety at 4-
position and chlorophenyl moiety at 2-position for exhibiting selective topo IIα inhibitory and
antiproliferative activity. Among 17-25, in general compounds with 2′-hydroxyl group at 2-
phenyl ring displayed weak topo IIα inhibition and antiproliferative activity, suggesting
introduction of 3′- and 4′-hydroxyl group is more important than 2′-hydroxyl group at 2-
phenyl ring for strongly inhibiting topo IIα activity and antiproliferative activity. However,
selective topo IIα inhibitory activity and antiproliferative activity might not be correlated to
the position of chlorine substitution on 4-phenyl ring.
Furthermore, to investigate the effect of ring expansion, in this study, we investigated
structure-activity relationships of prepared hydroxy and chloro-substituted 2,4-diphenyl 5H-
chromeno[4,3-b]pyridines, and previously reported corresponding benzofuro[3,2-
2
3
b]pyridines. Table 2 displays the relative topo I and IIα inhibitory potencies of the
synthesized compounds 8-16 and corresponding benzofuro[3,2-b]pyridines A-I at 100 µM
and 20 µM concentrations. From comparison of relative topo inhibitory potency, it was
evident that the replacement of five membered heterocyclic ring moiety with six membered
heterocyclic ring moiety significantly influenced the topo I and IIα inhibitory properties of
compounds. Compounds A-I having benzofuropyridine skeleton displayed dual topo I and
IIα inhibitory activity, while currently modified compounds possessing chromenopyridine
moiety have showed selective topo IIα inhibition. The overall SAR study is illustrated in
Figure 4.
1
5

Figure 4. Overall structure-activity relationships of hydroxy and chloro-substituted 2,4-
diphenyl 5H-chromeno[4,3-b]pyridines.
1
6

Table 2. Relative topo I and IIα inhibitory potencies of synthesized compounds compared to camptothecin and etoposide.
Relative potency* for topo I and IIα (% inhibition) of compounds compared to positive controls, camptothecin (topo I) and
etoposide (topo IIα)
Topo IIα
Topo I
Topo IIα
Topo I
100 µM
Reported a
Compound
Newly
Synthesized
Compound
1
00 µM 20 µM
100 µM 20 µM
100 µM 20 µM
20
µM
NT
NT
NT
0.05
NA
NT
NT
NT
NT
Substitution
b
R = 2-Cl, R = 2-OH
A
B
C
D
E
F
0.98
0.97
0.99
0.95
0.98
1.01
1.07
1.01
1.15
0.62
0.91
0.18
1.01
1.39
1.22
1.12
1.32
1.39
1.56
1.62
NT
8
1.19
1.13
1.34
1.37
1.26
1.07
1.24
1.44
1.32
1.33
1.46
1.59
1.61
1.79
1.80
2.10
1.61
1.18
0.06
0.06
0.10
0.99
0.36
0.36
NA
1
2
R = 3-Cl, R = 2-OH
0.38
0.93
0.98
2.02
2.09
1.99
1.29
1.40
9
1
2
R = 4-Cl, R = 2-OH
10
11
12
13
14
15
16
1
2
0.63
R = 2-Cl, R = 3-OH
0.42
1
2
R = 3-Cl, R = 3-OH
1
2
0.65
0.27
0.56
R = 4-Cl, R = 3-OH
1
2
R = 2-Cl, R = 4-OH
G
H
I
1
2
R = 3-Cl, R = 4-OH
NA
1
2
0.77
1.75
R = 4-Cl, R = 4-OH
NA
1
2
*
Relative potency: % inhibition of compound / % inhibition of positive control, NT: not tested, NA: not active.
Previously reported compounds A-I, ; newly synthesized compounds 8-16.
a
23 b
1
7

3
. Conclusion
In conclusion, we synthesized a new series of hydroxy and chloro-substituted 2,4-
diphenyl 5H-chromeno[4,3-b]pyridines based on the strategy of ring expansion from
constrained five membered heterocyclic ring to more stable six membered heterocycles. We
evaluated the influence of this replacement on topo I and IIα inhibitory activity, and
antiproliferative activity. All the synthesized compounds displayed selective topo IIα
inhibitory activity in contrast to dual topo I and IIα inhibitory activity of previously reported
benzofuro[3,2-b]pyridines. Most of the compounds showed comparatively better
antiproliferative activity against human cervix tumor cell line (HeLa) than etoposide.
Structure-activity relationships of compounds suggests the importance of hydroxyphenyl
moiety at 4-position of central pyridine ring for selectively inhibiting topo IIα activity and
antiproliferativ activity. Similarly, 3′- and 4′-hydroxyphenyl substitution was favorable for
better activity as compared to 2′-hydroxyphenyl substitution at 2- position of central pyridine.
Experiments to investigate mode of action of a compound 14, which showed potent and
selective topo IIα inhibition and strong antiproliferative activity, is underway and will be
reported in somewhere else. The findings from this study could be useful for researcher to
further design and develop new chromenopyridine analogues as safer and more efficacious
selective topo IIα targeting anticancer agents.
1
8

4
. Experimental
Commercially available starting materials and reagents were purchased from Sigma-
Aldrich, TCI Chemicals, Alfa-Aesar, and Junsei, and were used without further purification.
HPLC grade acetonitrile (ACN) and methanol were purchased from Burdick and Jackson,
USA. Column chromatography was carried out using silica gel (Kieselgel 60, 230-400 mesh,
Merck). Thin layer chromatography (TLC) was performed on silica gel plates (Kieselgel 60
F254, Merck) having layer thickness of 0.25 mm. Melting points were recorded using open
capillary tube on electrothermal 1A 9100 digital melting point apparatus and were
1
13
uncorrected. H NMR (250 MHz) and C NMR (62.5 MHz) spectra were recorded in Bruker
AMX 250 (250 MHz, FT) spectrometer using CDCl and DMSO-d as solvent, and
3
6
tetramethylsilane (TMS) as internal standard. All the chemical shifts values (δ) were recorded
in parts per million (ppm) and coupling constants (J) in hertz (Hz).
HPLC analyses were performed using two Shimadzu LC-10AT pumps gradient-
controlled HPLC system equipped with Shimadzu system controller (SCL-10A VP) and
photo diode array detector (SPD-M10A VP) utilizing Shimadzu LC Solution program.
®
Sample volume of 10 μL, was run in Waters X- Terra 5 μM reverse-phase C column (4.6
1
8
ⅹ
250 mm) with a gradient elution of 85% to 100% of B in A for 10 min followed by 100%
to 85% of B in A for 10 min at a flow rate of 0.7 mL/min at 254 nm UV detection, where
mobile phase A was doubly distilled water with 50 mM ammonium formate (AF) and B was
1
00% ACN. Purity of compound is stated as percent (%) and retention time in minutes. Mass
®
spectra of the compounds were recorded with Advion Expression CMS ESI-MS
spectrometer (Advion, Ithaca, NY, USA), using methanol: water: formic acid (80: 20: 0.1) as
a mobile phase.
1
9

4
. 1. General method for preparation of 4-chromanone intermediate 5a-5f
4
.1.1. Synthesis of 3-(2-hydroxybenzylidene)chroman-4-one (5a)
To the mixture of 2-hydroxybenzaldehyde 1a (1.6 mL, 15.0 mmol, 1.0 equiv.) and DIPEA
(
7.8 mL, 45.0 mmol, 3.0 equiv.) in CH Cl (25 mL) was added MOM-Cl (1.7 mL, 22.5 mmol,
2 2
1
.5 equiv.). The mixture was stirred for 3 h at room temperature. After the completion of
reaction monitored by TLC, H O (150 mL) was added and extracted with CH Cl (50 mL, 3
2
2
2
times). The organic layer was dried over anhydrous MgSO , filtered and concentrated in
4
vacuo at 60 °C and dried under high vacuum to give ayellowish brown oil. Silica gel column
chromatography was used to purify the compound using ethyl acetate and n-hexane as eluents
to yield 2a (2.40 g, 96.5%, 14.5 mmol) as a light yellow oil. In a further step, to the solution
of 2a (2.4 g, 14.5 mmol, 1.0 equiv.) and 4-chromanone 3 (2.7 g, 16.2 mmol, 1.0 equiv.) in
methanol (30 mL) was added aqueous NaOH (0.8 g in 11 mL H O) dropwise and stirred for
2
2
4 h at room temperature. The mixture was evaporated, extracted with ethyl acetate (200 mL),
and washed with distilled water (50 mL, 5 times) followed by saturated NaCl solution (50 mL,
times). The organic layer was dried with anhydrous MgSO and filtered off. Purification
2
4
was performed by silica gel column chromatography using ethyl acetate and n-hexane as
eluents to obtain 4a (4.59 g, 95.6%, 15.5 mmol) as a yellow solid. Finally, deprotection of 4a
was achieved by refluxing 4a (4.6 g, 15.5 mmol, 1.0 equiv.) with aqueous 2 M HCl (11.7 mL,
1
.5 equiv.) in ethanol (60 mL) for 1 h at 80 °C. The mixture was evaporated and extracted
with ethyl acetate (200 mL), washed with distilled water (50 mL, 4 times), and saturated
NaCl solution (50 mL). The organic layer was dried over anhydrous MgSO and filtered off.
4
Further purification was performed using silica gel column chromatography by elution of
ethyl acetate and n-hexane to give 4-chromanone intermediate 5a (1.96 g, 50.2%, 7.8 mmol)
as a yellow solid.
TLC (ethyl acetate/n-hexane = 1:2) R = 0.23, mp: 187.2-187.9 °C.
f
2
0

1
H NMR (250 MHz, DMSO-d ) δ 10.24 (s, 1H, phenyl 2-OH), 7.88-7.85 (m, 2H,
6
chromanone H-5 and =CH-), 7.58 (td, J = 6.90, 0.77 Hz, 1H, chromanone H-7), 7.28 (t, J =
7
8
1
1
1
.37 Hz, 1H, phenyl H-4), 7.14-7.08 (m, 2H, chromanone H-6 and phenyl H-6), 7.04 (d, J =
.30 Hz, 1H, chromanone H-8), 6.94 (d, J = 8.17 Hz, 1H, phenyl H-3), 6.88 (t, J = 7.47 Hz,
1
3
H, phenyl H-5), 5.30 (s, 2H, -CH -). C NMR (62.5 MHz, DMSO-d ) δ 181.46, 160.75,
2
6
56.89, 136.20, 133.17, 131.64, 130.63, 129.62, 127.33, 122.02, 121.70, 120.94, 119.11,
17.99, 115.83, 67.79.
4
.1.2. Synthesis of 3-(3-hydroxybenzylidene)chroman-4-one (5b)
To the mixture of 3-hydroxybenzaldehyde 1b (3.1 mL, 25.0 mmol, 1.0 equiv.) and DIPEA
13.1 mL, 75.0 mmol, 3.0 equiv.) in CH Cl (40 mL) was added MOM-Cl (2.9 mL, 37.5
(
2
2
mmol, 1.5 equiv.). The mixture was stirred for 3 h at room temperature. After the completion
of reaction monitored by TLC, H O (150 mL) was added and extracted with CH Cl (50 mL,
2
2
2
3
times). The organic layer was dried over anhydrous MgSO , filtered and concentrated in
4
vacuo at 60 °C and dried under high vacuum to afford alight brown oil. Silica gel column
chromatography was used to purify the compound using ethyl acetate and n-hexane as eluents
to yield 2b (3.59 g, 86.3%, 21.6 mmol) as a light yellow oil. In further step, to the solution of
2
b (3.6 g, 21.6 mmol, 1.0 equiv.) and 4-chromanone 3 (3.2 g, 21.6 mmol, 1.0 equiv.) in
methanol (45 mL) was added aqueous NaOH (1.0 g in 15 mL H O) dropwise and stirred for
2
2
4 h at room temperature. The mixture was evaporated, extracted with ethyl acetate (200 mL),
and washed with distilled water (50 mL, 6 times) followed by saturated NaCl solution (50 mL,
times). The organic layer was dried with anhydrous MgSO and filtered off. Purification
2
4
was performed by silica gel column chromatography using ethyl acetate and n-hexane as
eluents to obtain 4b (4.73 g, 74.0%, 15.9 mmol) as a yellow solid. Finally, deprotection of 4b
was achieved by refluxing 4b (4.7 g, 15.9 mmol, 1.0 equiv.) with aqueous 2 M HCl (11.9 mL,
2
1

1
.5 equiv.) in ethanol (70 mL) for 3.5 h at 80 °C. Then, the reaction mixture was evaporated
and extracted with ethyl acetate (200 mL), washed with distilled water (50 mL, 4 times), and
saturated NaCl solution (50 mL). The organic layer was dried over anhydrous MgSO and
4
filtered off. Further purification was performed using silica gel column chromatography by
elution of ethyl acetate and n-hexane to give 4-chromanone intermediate 5b (2.03 g, 50.5%,
8
.1 mmol) as a yellow solid.
TLC (ethyl acetate/ n -hexane = 1:3) R = 0.28, mp: 219.3-219.8 °C.
f
1
H NMR (250 MHz, DMSO-d ) δ 9.68 (s, 1H, phenyl 3-OH), 7.87 (dd, J = 7.85, 1.67 Hz, 1H,
6
chromanone H-5), 7.65 (br s, 1H, -CH-), 7.58 (td, J = 7.05 1.77 Hz, 1H, chromanone H-7),
7
.28 (t, J = 7.82 Hz, 1H, phenyl H-5), 7.12 (td, J = 7.95, 1.0 Hz, 1H, chromanone H-6), 7.04
(dd, J = 8.32, 0.55 Hz, 1H, chromanone H-8), 6.88-6.81 (m, 3H, phenyl H-2, H-4, and H-6),
1
3
5
1
6
.38 (s, 2H, -CH -). C NMR (62.5 MHz, DMSO-d ) δ 181.15, 160.60, 157.47, 136.69,
2 6
36.16, 134.93, 130.56, 129.81, 127.20, 121.90, 121.45, 121.05, 117.86, 116.85, 116.65,
7.33.
4
.1.3. Synthesis of 3-(4-hydroxybenzylidene)chroman-4-one (5c)
To a solution of 4-chromanone 3 (1.5 g, 10.0 mmol, 1.0 equiv.) and 4-hydroxybenzaldehyde
c (1.2 g, 10.0 mmol, 1.0 equiv.) in dioxane (10 mL) was slowly added boron trifluoride
1
diethyl etherate (2.1 mL, 15.0 mmol, 1.5 equiv.). The mixture was stirred for 24 h at room
temperature. After the completion of reaction monitored by TLC, the mixture was diluted
with ethyl acetate (100 mL) and washed with distilled water (40 mL, 4 times), and saturated
NaCl (40 mL, 2 times). The organic layer was dried with anhydrous MgSO and filtered off.
4
Silica gel column chromatography was used to purify the compound using ethyl acetate and
n-hexane as eluent to afford 4-chromaonne intermediate 5c (1.43 g, 56.6%, 5.6 mmol) as a
yellow solid.
2
2

TLC (ethyl acetate/ n -hexane = 1:2) R = 0.23, mp: 238.9-239.6 °C.
f
1
H NMR (250 MHz, DMSO-d ) δ 10.18 (s, 1H, phenyl 4-OH), 7.85 (dd, J = 7.82, 1.62 Hz,
6
1
7
7
5
1
H, chromanone H-5), 7.66 (br s, 1H, -CH-), 7.56 (td, J = 8.50, 1.67 Hz, 1H, chromanone H-
), 7.33 (d, J = 8.62 Hz, 2H, phenyl H-2 and H-6), 7.10 (t, J = 7.37 Hz, 1H, chromanone H-6),
.03 (d, J = 8.25 Hz, 1H, chromanone H-8), 6.87 (d, J = 8.55 Hz, 2H, phenyl H-3 and H-5),
13
.41 (s, 2H, -CH -). C NMR (62.5 MHz, DMSO-d ) δ 181.13, 160.52, 159.40, 137.06,
2
6
36.06, 132.93 (2C), 127.65, 127.28, 124.87, 121.97, 121.71, 117.91, 115.88 (2C), 67.65.
4
.1.4. Synthesis of 3-(2-chlorobenzylidene)chroman-4-one (5d)
To the solution of 4-chromanone 3 (0.9 g, 6.0 mmol, 1.0 equiv.) and 2-chlorobenzaldehyde
d (1.0 mL, 9.0 mmol, 1.5 equiv.) in ethanol (6 mL), 5% aqueous NaOH solution (4 mL) was
1
added dropwise which resulted precipitation. The mixture was stirred for 1 h at room
temperature. The precipitate was isolated by filtration and washed with cold ethanol to obtain
4
-chromanone intermediate 5d (1.27 g, 78.2%, 4.7 mmol) as a white solid.
o
TLC (ethyl acetate/n-hexane = 1:7 v/v) R = 0.27; mp: 114.5-115.1 C
f
1
H NMR (250 MHz, CDCl ) δ 8.02 (dd, J = 7.87, 1.70 Hz, 1H, chromanone H-5), 7.94 (s, 1H,
3
=CH-), 7.51-7.44 (m, 2H, chromanone H-6 and 2-phenyl H-6), 7.37-7.27 (m, 2H, 2-phenyl
H-4 and H-5), 7.12-7.03 (m, 2H, chromanone H-7 and 2-phenyl H-3), 6.95 (dd, J = 8.35, 0.67
1
3
Hz, 1H, chromanone H-8), 5.16 (s, 2H, chromanone H-2). C NMR (62.5 MHz, CDCl ) δ
3
1
1
82.35, 161.47, 136.28, 135.15, 134.77, 133.04, 132.59, 130.74, 130.54, 130.27, 128.21,
26.83, 122.22, 122.11, 118.19, 67.70.
2
3

4
.1.5. Synthesis of 3-(3-chlorobenzylidene)chroman-4-one (5e)
The procedure described in Section 4.1.4 was employed with 3 (0.9 g, 6.0 mmol, 1.0 equiv.),
1
5
e (1.0 mL, 9.0 mmol, 1.5 equiv.) and aqueous NaOH solution (4 mL) to obtain intermediate
e (1.33 g, 81.8%, 4.9 mmol) as a white solid.
o
TLC (ethyl acetate/n-hexane = 1:10 v/v) R = 0.26; mp: 138.9-139.7 C
f
1
H NMR (250 MHz, CDCl ) δ 8.00 (dd, J = 7.90, 1.67 Hz, 1H, chromanone H-5), 7.76 (s, 1H,
3
=CH-), 7.48 (td, J = 7.12, 1.77 Hz, 1H, chromanone H-6), 7.37-7.32 (m, 2H, 2-phenyl H-5
and H-6), 7.26 (br, 1H, 2-phenyl H-2), 7.19-7.14 (m, 1H, 2-phenyl H-4), 7.06 (td, J = 7.07,
1
2
1
.02 Hz, 1H, chromanone H-7), 6.95 (dd, J = 8.32, 0.65 Hz, 1H, chromanone H-8), 5.28 (s,
1
3
H, chromanone H-2). C NMR (62.5 MHz, CDCl ) δ 182.13, 161.34, 136.29, 135.93,
3
34.93, 132.26, 130.20, 129.82, 129.62, 128.17, 128.15, 122.26, 122.04, 118.18, 67.57.
4
.1.6. Synthesis of 3-(4-chlorobenzylidene)chroman-4-one (5f)
The procedure described in Section 4.1.4 was employed with 3 (0.9 g, 6.0 mmol, 1.0 equiv.),
f (1.3 g, 9.0 mmol, 1.5 equiv.) and aqueous NaOH solution (4 mL) to obtain intermediate 5f
0.99 g, 61.3%, 3.7 mmol) as a white solid.
1
(
o
TLC (ethyl acetate/n-hexane = 1:10 v/v) R = 0.33; mp: 128.8-129.3 C
f
1
H NMR (250 MHz, CDCl ) δ 7.99 (dd, J = 7.87, 1.75 Hz, 1H, chromanone H-5), 7.78 (s, 1H,
3
=CH-), 7.51-7.38 (m, 3H, chromanone H-6, 2-phenyl H-2 and H-6), 7.22 (d, J = 8.65 Hz, 2H,
2
0
-phenyl H-3 and H-5), 7.05 (td, J = 7.80, 1.02 Hz, 1H, chromanone H-7), 6.95 (dd, J = 8.35,
1
3
.60 Hz, 1H, chromanone H-8), 5.29 (s, 2H, chromanone H-2). C NMR (62.5 MHz, CDCl )
3
δ 182.20, 161.26, 134.26, 136.24, 135.77, 132.95, 131.54, 131.40 (2C), 129.25 (2C), 128.15,
22.24, 122.07, 118.14, 67.62.
1
2
4

4
. 2.General method for preparation of aryl pyridinium iodide salts 7 (R =a-f)
Aryl pyridinium iodide salts 7 (R = a-f) were synthesized by refluxing aryl methyl ketones 6
R = a-f) with the equivalent amount of iodine in pyridine at 140 °C for 3 hours. The mixture
(
was cooled to room temperature which resulted precipitation. The precipitate was filtered and
washed with cold pyridine followed by drying overnight to yield 52.2-93.0% of 7a-7f.
Compounds were used without further purification.
4
. 3. General method for preparation of 8-25
Modified Kröhnke pyridine synthetic method was used for the synthesis of chloro and
hydroxy-substituted 2,4-diphenyl 5H-chromeno[4,3-b]pyridines (8-25). To the mixture of
chromanone intermediates (5a-5f) and pyridinium iodide salts 7 (R = a-f) in glacial acetic
acid was added anhydrous ammonium acetate (10.0 equiv.). The mixtue was refluxed at
1
00 °C for 12-24 h. After the completion of reaction monitored by TLC, the mixture was
extracted with ethyl acetate then washed with distilled water and saturated NaCl solution. The
organic layer was dried with anhydrous MgSO and filtered off. Further purification was
4
performed by silica gel column chromatography using ethyl acetate and n-hexane as eluents
to afford 2,4-disubstituted 5H-chromeno[4,3-b]pyridines (8-25) with the yields of 22.1-74.3%.
4
.3.1. Synthesis of 2-(2-(2-chlorophenyl)-5H-chromeno[4,3-b]pyridin-4-yl)phenol (8)
The procedure described in Section 4.3 was employed with 5a (0.3 g, 1.0 mmol, 1.0 equiv.),
a (0.5 g, 1.5 mmol, 1.5 equiv.), and dry ammonium acetate (0.8 g, 10.0 mmol, 10.0 equiv.)
7
in glacial acetic acid (3 mL) to afford 8 (0.10 g, 26.8%, 0.3 mmol) as a white solid.
o
TLC (ethyl acetate/n-hexane = 1:3 v/v) R = 0.27; mp: 208.1-208.9 C; HPLC: Retention time:
f
+
1
1.7 min, purity: 96.6%; ESI-MS: m/z calcd for C H ClNO [M+H] : 386.09; found: 386.16.
2
4
16
2
2
5

1
H NMR (250 MHz, CDCl ) δ 8.35 (dd, J = 7.72, 1.55 Hz, 1H, chromeno H-5), 7.77 (dd, J =
3
7
2
.20, 2.12 Hz, 1H, 2-phenyl H-6), 7.55 (s, 1H, pyridine H-3), 7.48 (dd, J = 7.60, 1.50 Hz, 1H,
-phenyl H-3), 7.41-7.27 (m, 4H, chromeno H-7, 2-phenyl H-4, H-5 and 4-phenyl H-5), 7.19
(dd, J = 7.57, 1.60 Hz, 1H, 4-phenyl H-6), 7.11-7.01 (m, 2H, chromeno H-6 and 4-phenyl H-
4
4
1
1
), 6.95 (d, J = 8.12 Hz, 2H, chromeno H-8 and 4-phenyl H-3), 5.13 (s, 2H, chromeno H-2),
1
3
.98 (s, 1H, 4-phenyl 2-OH). C NMR (62.5 MHz, CDCl ) δ 156.56, 156.10, 152.36, 149.17,
3
41.74, 138.83, 132.30, 131.91, 131.38, 130.49, 130.30, 130.23, 129.70, 127.03, 125.36,
24.86, 123.81, 123.67, 123.47, 122.33, 121.23, 116.80, 116.14, 66.05.
4
.3.2. Synthesis of 2-(2-(3-chlorophenyl)-5H-chromeno[4,3-b]pyridin-4-yl)phenol (9)
The procedure described in Section 4.3 was employed with 5a (0.3 g, 1.0 mmol, 1.0 equiv.),
b (0.5 g, 1.5 mmol, 1.5 equiv.), and dry ammonium acetate (0.8 g, 10.0 mmol, 10.0 equiv.)
7
in glacial acetic acid (3 mL) to afford 9 (0.09 g, 24.4%, 0.2 mmol) as a yellow solid.
o
TLC (ethyl acetate/n-hexane = 1:3 v/v) R = 0.29; mp: 181.7-182.3 C; HPLC: Retention time:
f
+
1
1.8 min, purity: 95.4%; ESI-MS: m/z calcd for C H ClNO [M+H] : 386.09; found: 386.28.
2
4
16
2
1
H NMR (250 MHz, CDCl ) δ 8.41 (dd, J = 7.71, 1.65 Hz, 1H, chromeno H-5), 7.79 (dd, J =
3
7
.21, 2.22 Hz, 1H, 2-phenyl H-6), 7.58 (s, 1H, pyridine H-3), 7.48 (dd, J = 7.65, 1.49 Hz, 1H,
2
-phenyl H-3), 7.38-7.10 (m, 4H, chromeno H-7, 2-phenyl H-4, H-5 and 4-phenyl H-5), 7.13
(dd, J = 7.57, 1.60 Hz, 1H, 4-phenyl H-6), 7.01-6.89 (m, 2H, chromeno H-6 and 4-phenyl H-
4
4
1
1
), 6.95 (d, J = 8.12 Hz, 2H, chromeno H-8 and 4-phenyl H-3), 5.16 (s, 2H, chromeno H-2),
1
3
.99 (s, 1H, 4-phenyl 2-OH). C NMR (62.5 MHz, CDCl ) δ 156.51, 156.11, 152.36, 149.25,
3
41.74, 138.73, 132.30, 131.88, 131.37, 130.52, 130.30, 130.19, 129.70, 127.13, 125.36,
24.76, 123.81, 123.60, 123.47, 122.29, 121.28, 116.73, 116.34, 66.07.
2
6

4
.3.3. Synthesis of 2-(2-(4-chlorophenyl)-5H-chromeno[4,3-b]pyridin-4-yl)phenol (10)
The procedure described in Section 4.3 was employed with 5a (0.2 g, 0.8 mmol, 1.0 equiv.),
c (0.4 g, 1.2 mmol, 1.5 equiv.), and dry ammonium acetate (0.6 g, 8.0 mmol, 10.0 equiv.) in
7
glacial acetic acid (2 mL) to afford 10 (0.12 g, 30.9%, 0.3 mmol) as a white solid.
o
TLC (ethyl acetate/n-hexane = 1:3 v/v) R = 0.27; mp: 199.6-200.4 C; HPLC: Retention time:
f
+
7
.3 min, purity: 97.5%; ESI-MS: m/z calcd for C H ClNO [M+H] : 386.09; found: 386.19.
2
4
16
2
1
H NMR (250 MHz, CDCl ) δ 8.42 (dd, J = 7.7, 1.8 Hz, 1H, chromeno H-5), 8.08 (d, J = 8.6
3
Hz, 2H, 2-phenyl H-2 and H-6), 7.56 (s, 1H, pyridine H-3), 7.44 (d, J = 8.6 Hz, 2H, 2-phenyl
H-3 and H-5), 7.39-7.27 (m, 2H, chromeno H-7 and 4-phenyl H-5), 7.22-7.01 (m, 3H,
chromeno H-6, 4-phenyl H-4 and H-6), 6.95 (dd, J = 8.1, 1.1 Hz, 2H, chromeno H-8 and 4-
1
3
phenyl H-3), 5.09 (s, 2H, chromeno H-2), 4.99 (s, 1H, 4-phenyl 2-OH). C NMR (62.5 MHz,
CDCl ) δ 156.90, 155.51, 152.52, 149.26, 143.14, 137.62, 135.52, 131.68, 130.76, 130.45,
3
1
29.12 (2C), 128.44 (2C), 125.51, 124.14, 124.06, 123.70, 122.54, 121.56, 120.43, 117.11,
116.33, 66.29.
4
.3.4. Synthesis of 3-(2-(2-chlorophenyl)-5H-chromeno[4,3-b]pyridin-4-yl)phenol (11)
The procedure described in Section 4.3 was employed with 5b (0.3 g, 1.0 mmol, 1.0 equiv.),
a (0.5 g, 1.5 mmol, 1.5 equiv.), and dry ammonium acetate (0.8 g, 10.0 mmol, 10.0 equiv.)
7
in glacial acetic acid (3 mL) to afford 11 (0.18 g, 46.7%, 0.5 mmol) as a white solid.
o
TLC (ethyl acetate/n-hexane = 1:5 v/v) R = 0.19; mp: 217.8-218.4 C; HPLC: Retention time:
f
+
5
.7 min, purity: 98.4%; ESI-MS: m/z calcd for C H ClNO [M+H] : 386.09; found: 386.18.
2
4
16
2
1
H NMR (250 MHz, CDCl ) δ 8.33 (dd, J = 7.72, 1.62 Hz, 1H, chromeno H-5), 7.73 (dd, J =
3
7
.15, 2.22 Hz, 1H, 2-phenyl H-6), 7.51 (s, 1H, pyridine H-3), 7.47 (dd, J = 7.25, 1.97 Hz, 1H,
2
-phenyl H-3), 7.40-7.27 (m, 4H, chromeno H-7, 2-phenyl H-4, H-5 and 4-phenyl H-5), 7.09
(td, J = 7.55, 1.10 Hz, 1H, chromeno H-6), 6.95 (dd, J = 8.10, 0.90 Hz, 1H, chromeno H-8),
2
7

6
.89-6.85 (m, 2H, 4-phenyl H-4 and H-6), 6.77-6.76 (m, 1H, 4-phenyl H-2), 5.40 (s, 1H, 4-
1
3
phenyl 3-OH), 5.24 (s, 2H, chromeno H-2). C NMR (62.5 MHz, CDCl ) δ 156.56, 156.04,
3
1
1
56.01, 149.14, 146.29, 139.06, 138.87, 132.52, 132.06, 131.55, 130.43, 130.24, 129.92,
27.23, 125.58, 124.50, 123.47, 122.71, 122.46, 121.20, 116.99, 115.90, 115.71, 66.06.
4
.3.5. Synthesis of 3-(2-(3-chlorophenyl)-5H-chromeno[4,3-b]pyridin-4-yl)phenol (12)
The procedure described in Section 4.3 was employed with 5b (0.3 g, 1.0 mmol, 1.0 equiv.),
b (0.5 g, 1.5 mmol, 1.5 equiv.), and dry ammonium acetate (0.8 g, 10.0 mmol, 10.0 equiv.)
7
in glacial acetic acid (3 mL) to afford 12 (0.29 g, 74.3%, 0.7 mmol) as a yellowish white
solid.
o
TLC (ethyl acetate/n-hexane = 1:5 v/v) R = 0.20; mp: 230.9-231.5 C; HPLC: Retention time:
f
+
9
.4 min, purity: 97.4 %; ESI-MS: m/z calcd for C H ClNO [M+H] : 386.09; found: 386.21.
2
4
16
2
1
H NMR (250 MHz, DMSO-d ) δ 9.71 (s, 1H, 4-phenyl 3-OH), 8.36-8.22 (m, 3H, chromeno
6
H-5, 2-phenyl H-2 and H-6), 7.90 (s, 1H, pyridine H-3), 7.54-7.49 (m, 2H, 2-phenyl H-4 and
H-5), 7.43-7.31 (m, 2H, chromeno H-7, 4-phenyl H-5), 7.19 (td, J = 7.52, 0.95 Hz, 1H,
chromeno H-6), 7.01 (d, J = 7.37 Hz, 1H, chromeno H-8), 6.93-6.88 (m, 3H, 4-phenyl H-2,
1
3
H-4 and H-6), 5.27 (s, 2H, chromeno H-2). C NMR (62.5 MHz, DMSO-d ) δ 157.53,
6
1
1
6
56.01, 153.47, 147.83, 147.50, 140.36, 137.65, 133.75, 131.54, 130.58, 129.76, 129.01,
26.32, 125.31, 124.85, 123.03, 122.79, 122.32, 119.98, 119.27, 116.71, 115.76, 115.47,
5.36.
4
.3.6. Synthesis of 3-(2-(4-chlorophenyl)-5H-chromeno[4,3-b]pyridin-4-yl)phenol (13)
The procedure described in Section 4.3 was employed with 5b (0.3 g, 1.0 mmol, 1.0 equiv.),
c (0.5 g, 1.5 mmol, 1.5 equiv.), and dry ammonium acetate (0.8 g, 10.0 mmol, 10.0 equiv.)
7
2
8

in glacial acetic acid (3 mL) to afford 13 (0.24 g, 62.8%, 0.6 mmol) as a yellowish white
solid.
o
TLC (ethyl acetate/n-hexane = 1:5 v/v) R = 0.21; mp: 178.8-179.6 C; HPLC: Retention time:
f
+
8
.7 min, purity: 97.7%; ESI-MS: m/z calcd for C H ClNO [M+H] : 386.09; found: 386.26.
2
4
16
2
1
H NMR (250 MHz, CDCl ) δ 8.42 (d, J = 7.67 Hz, 1H, chromeno H-5), 8.07 (d, J = 8.52 Hz,
3
2
H, 2-phenyl H-2 and H-6), 7.53 (s, 1H, pyridine H-3), 7.43 (d, J = 8.50 Hz, 2H, 2-phenyl H-
and H-5), 7.36-7.30 (m, 2H, chromeno H-7 and 4-phenyl H-5), 7.14 (t, J = 7.57 Hz, 1H,
3
chromeno H-6), 6.97-6.83 (m, 3H, chromeno H-8, 4-phenyl H-4 and H-6), 6.75 (br s, 1H, 4-
1
3
phenyl H-2), 5.21 (br s, 3H, 4-phenyl 3-OH and chromeno H-2). C NMR (62.5 MHz,
CDCl ) δ 156.61, 156.01, 155.12, 149.08, 147.30, 139.06, 137.56, 135.44, 131.63, 130.32,
3
1
6
29.09 (2C), 128.37 (2C), 125.48, 123.73, 122.60, 121.07, 119.76, 117.05, 115.9, 115.52,
6.05.
4
.3.7. Synthesis of 4-(2-(2-chlorophenyl)-5H-chromeno[4,3-b]pyridin-4-yl)phenol (14)
The procedure described in Section 4.3 was employed with 5c (0.2 g, 0.8 mmol, 1.0 equiv.),
a (0.4 g, 1.2 mmol, 1.5 equiv.), and dry ammonium acetate (0.6 g, 8.0 mmol, 10.0 equiv.) in
7
glacial acetic acid (2 mL) to afford 14 (0.14 g, 37.1%, 0.4 mmol) as a yellowish white solid.
o
TLC (ethyl acetate/n-hexane = 1:3 v/v) R = 0.26; mp: 231.4-232.2 C; HPLC: Retention time:
f
+
6
.5 min, purity: 96.6%; ESI-MS: m/z calcd for C H ClNO [M+H] : 386.09; found: 386.31.
2
4
16
2
1
H NMR (250 MHz, DMSO-d ) δ 9.90 (s, 1H, 4-phenyl 4-OH), 8.17 (dd, J = 7.70, 1.40 Hz,
6
1
H, chromeno H-5), 7.76-7.72 (m, 1H, 2-phenyl H-6), 7.62-7.58 (m, 1H, 2-phenyl H-3),
.51-7.46 (m, 3H, pyridine H-3, chromeno H-7 and 2-phenyl H-4), 7.41-7.29 (m, 3H, 2-
7
phenyl H-5, 4-phenyl H-2 and H-6), 7.12 (t, J = 7.60 Hz, 1H, chromeno H-6), 7.01 (d, J =
.05 Hz, 1H, chromeno H-8), 6.91 (d, J = 8.42 Hz, 2H, 4-phenyl H-3 and H-5), 5.34 (s, 2H,
8
2
9